Chemically modified lipolytic enzyme

ABSTRACT

Lipolytic enzymes are chemically modified by covalently linking one or more (particularly 1-3) hydrophobic groups to the enzyme molecule. The chemical modification improves the performance of the lipolytic enzyme, e.g., in baking or in detergents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/019,156,filed Dec. 3, 2001, which is a 35 U.S.C. 371 national application ofPCT/DK00/00300 filed Jun. 2, 2000 (the international application waspublished under PCT Article 21(2) in English) and claims, under 35U.S.C. 119, priority of Danish application no. PA 1999 00778 filed Jun.2, 1999 and the benefit of U.S. provisional application no. 60/138,081filed Aug. 6, 1999, the contents of which are fully incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to a chemically modified lipolytic enzyme,its preparation and its to uses thereof.

BACKGROUND OF THE INVENTION

Lipolytic enzymes such as lipases and phospholipases are used, e.g., indetergents and baking.

Thus, lipases have been used for a number of years as detergent enzymesto remove lipid or fatty stains from clothes and other textiles,particularly a lipase derived from Humicola lanuginosa (EP 258 068 andEP 305 216) sold under the trade name Lipolase® (product of Novo NordiskA/S).

Fatty acid-modified lipases and their use in transesterification havebeen described. M. Murakami et al., JAOCS, 70 (6), 571-574 (1993); K.Green et al., JAOCS, 75 (11), 1519-1526 (1998).

It is also known to add lipases and phospholipases to breadmaking dough.WO 94/04035; WO 98/26057.

SUMMARY OF THE INVENTION

The inventors have developed lipolytic enzymes which are chemicallymodified by covalently linking one or more hydrophobic groups to theenzyme molecule. They found that the chemical modification may improvethe performance of the lipolytic enzyme, e.g., in baking or indetergents. The benefits may include improved thermostability and analtered substrate specificity. A modified lipase or cutinase may showimproved detergency, particularly improved first-wash performance,whiteness maintenance, dingy cleanup, and reduced formation of fattyacids during the drying process with less risk of forming an unpleasantsmell. The benefits in baking include an increased loaf volume.

Accordingly, the invention provides a lipolytic enzyme which ischemically modified by having one or more (particularly 1-3) hydrophobicgroups covalently linked to the enzyme. The invention also provides usof such modified lipolytic enzyme in detergents and baking.

The invention further provides a method of preparing a chemicallymodified lipolytic enzyme by covalently linking hydrophobic groups to aparent lipolytic enzyme. Optionally, the amino acid sequence of theenzyme may be modified before the covalent linking.

DETAILED DESCRIPTION OF THE INVENTION

Parent Lipolytic Enzyme

The lipolytic enzyme is an enzyme classified under the EnzymeClassification number E.C. 3.1.1.—(Carboxylic Ester Hydrolases) inaccordance with the Recommendations (1992) of the International Union ofBiochemistry and Molecular Biology (IUBMB). Thus, the lipolytic enzymemay exhibit hydrolytic activity, typically at a water/lipid interface,towards carboxylic ester bonds in substrates such as mono-, di- andtriglycerides, phospholipids, thioesters, cholesterol esters,wax-esters, cutin, suberin, synthetic esters or other lipids mentionedin the context of E.C. 3.1.1. The lipolytic enzyme may, e.g., haveactivity lipase activity (with triglycerides as substrate),phospholipase activity, esterase activity or cutinase activity.

The parent lipolytic enzyme may be prokaryotic, particularly a bacterialenzyme, e.g. from Pseudomonas. Examples are Pseudomonas lipases, e.g.from P. cepacia, P. glumae, P. pseudoalcaligenes and Pseudomonas sp.strain SD 705. Other examples are bacterial cutinases, e.g. fromPseudomonas such as P. mendocina (U.S. Pat. No. 5,389,536) or P. putida(WO 88/09367).

Alternatively, the parent lipolytic enzyme may be eukaryotic, e.g.fungal, such as lipolytic enzymes of the Humicola family and theZygomycetes family and fungal cutinases. Examples of fungal cutinasesare the cutinases of Fusarium solani pisi and Humicola insolens.

The Humicola family of lipolytic enzymes consists of the lipase from H.lanuginosa strain DSM 4109 and lipases having more than 50% homologywith said lipase. The lipase from H. lanuginosa (synonym Thermomyceslanuginosus) is described in EP 258 068 and EP 305 216, and has theamino acid sequence shown in positions 1-269 of SEQ ID NO: 2 of U.S.Pat. No. 5,869,438.

The Humicola family also includes the following lipolytic enzymes:lipase from Penicillium camembertdi, lipase/phospholipase from Fusariumoxysporum, lipase from F. heterosporum, lysophospholipase fromAspergillus foetidus, phospholipase A1 from A. oryzae, lipase from A.oryzae, lipase/ferulic acid esterase from A. niger, lipase/ferulic acidesterase from A. tubingensis, lipase from A. tubingensis,lysophospholipase from A. niger and lipase from F. solani.

The Zygomycetes family comprises lipases having at least 50% homologywith the lipase of Rhizomucor miehei. This family also includes thelipases from Absidia reflexa, A. sporophora, A. corymbifera, A.blakesleeana, A. griseola and Rhizopus oryzae.

The phospholipase may have A₁ or A₂ activity to remove fatty acid fromthe phospholipid and form a lyso-phospholipid, or it may be havephospholipase B or lysophospholipase activity. It may or may not havelipase activity, i.e. activity on triglycerides. The phospholipase maybe of animal origin, e.g. from pancreas (e.g. bovine or porcinepancreas), snake venom or bee venom. Alternatively, the phospholipasemay be of microbial origin, e.g. from filamentous fungi, yeast orbacteria, such as the genus Aspergillus, Fusarium or Hyphozyma (WO98/18912), particularly the species A. niger or F. oxysporum (WO98/26057).

Other examples of lipolytic enzymes are described in PCT/DK 99/00664(Danish patent application PA 1998 01572).

The lipolytic enzyme may be native to such source, or it may be avariant thereof obtained by altering the amino acid sequence. Examplesof such variants are those described in WO 92/05249, WO 94/25577, WO95/22615, WO 97/04079 and WO 97/07202, WO 98/08939, PCT/DK 99/00068, EP99610010.3 and Danish patent application PCT/DK 00/00156 (PA 199900441). A specific example is a variant of the Humicola lanuginosalipase having the mutations E1SPPCGRRP+E99N+N101S+E239C+Q249R.

Hydrophobic Group

Generally, a hydrophobic group can be identified from a negativefree-energy-of-transfer from water to oil. More specifically, suitablehydrophobic groups can be identified in a partition coefficientexperiment where the two media are an aqueous detergent solution and asurface containing the (lipid) substrate of choice. The general conceptis described in standard text books such as C. Tanford (1980), Thehydrophobic effect, Wiley, N.Y.

The hydrophobic group may be a fatty acyl group, particularly having12-22 or 14-20 carbon atoms, straight-chain or branched, saturated,mono- or polyunsaturated, optionally substituted. Examples are myristoyl(tetradecanoyl), palmitoyl (hexadecanoyl), stearoyl (octadecanoyl) andarachidoyl (eicosanoyl).

Other examples of hydrophobic groups are those commonly found insurfactants, e.g. a hydrophobic polymer group such as poly-alkoxy oralkyl-polyalkoxy of the general formula R¹—(O—CHR²—CH₂)_(n) wherein R¹is H or C₁₄-C₂₂ alkyl, R² is H or methyl, and n is 10-200, e.g. 20-100.

The hydrophobic group(s) may particularly be linked to an amino acid inthe lipid contact zone of the lipolytic enzyme (as described in WO92/05249) or within 5 Å from the edge of said zone.

The modified lipolytic enzyme containing one, two or three hydrophobicgroups will be referred to as a monopod, dipod or tripod, respectively.

Covalent Linking

The hydrophobic group may be covalently linked, e.g., to an amino group(lysine or N-terminal), a thiol group (cysteine residues), a hydroxylgroup (serine or threonine) or a carboxyl group (glutamic acid, asparticacid or C-terminal) in the amino acid sequence of the lipolytic enzyme.The covalent linking can be done by methods known in the art.

Thus, linking to amino groups can be done through a reactiveintermediate such as an N-hydroxy-succinimide activated fatty acids,e.g. stearoyl or arachidoyl acid N-hydroxy-succinimide, or maleimideesters at high pH (e.g. pH 8-9).

Linking to a thiol group can be done by linking to a maleimide ester atpH 6.5-7, by reaction with fatty acid methane thiosulfonate (e.g. at pH8), or as described in |WO 91/16423|, |WO 98/23732 or WO 99/3732|.

Linking to a carboxyl group can be done by linking a hydrophobic amineas described in |WO 95/09909|.

To ensure that the number of hydrophobic groups linked to each enzymemolecule will be from one to three, one strategy uses a lipolytic enzymehaving an amino acid sequence with one, two or three of the group inquestion (e.g. amino or thiol). This is discussed below.

Another strategy is to choose the conditions (amounts of reagents etc.)for the linking reaction such that, on average, 1-3 hydrophobic groupswill be linked to each enzyme molecule.

Amino Acid Sequence

A lipolytic enzyme with 1-3 groups may be a variant obtained bymodifying the amino acid sequence of a given lipolytic enzyme byrecombinant technology using site-directed mutagenesis.

Thiol groups can also be introduced by chemical reaction as described inDuncan et al., (1983) Anal. Biochem.132, 68-73.

The N-terminal amino group may be eliminated by using site-directedmutagenesis to change the N-terminal to glutamine and after expressionconvert this to pyroglutamate by cyclization (Thiede, B, Lamer S.,Mattow J., Siejak F., Dimmler C., Rudel T., Jungblut P R.; rapidcommunications in Mass spectroscopy Vol 14 (6) pp. 496-502 (2000). Achoice for expression of pyroglutamate containing peptide in filamentousfungi, could be to use parts of the signal peptide and N-terminal of theperoxidase from the filamentous fungi Coprinus cinereus. This peroxidasehas an N-terminal pyroglutamate (Baunsgaard L., Dalboge H., Houen G.,Rasmussen E M, Welinder K G:, European journal of Biochemistry vol. 213(1) 605-611 (1993).

The peroxidase N-terminal and part of the neighboring amino acids can beconferred to the N-terminal of the lipolytic enzyme by standardmolecular biological techniques to created a variant with apyro-glutamic N-terminal.

Lipolytic Enzyme Variant

The lipolytic enzyme variant may be designed to change the number andlocation of amino or thiol groups by amino acid insertion, deletionand/or substitution involving lysine or cysteine.

A change in the number of lysine residues may be balanced by a change inthe number of other charged amino acids may, to keep the isoelectricpoint fairly unchanged. Thus, lysine may be substituted with anotherpositively charged amino acid (histidine or arginine).

One strategy is to remove some of the lysine residues by substitution ordeletion and keep 1-3 lysine residues unchanged. Thus, of the 6 lysineresidues in the Humicola lanuginosa lipase, one or more of the followingmay be retained: K24, K98, K233.

Another strategy is to remove all lysine residues in the nativelipolytic enzyme by substitution or deletion (and optionally remove theN-terminal amino group) and to introduce one, two or three lysineresidues by substitution or insertion at selected positions in the lipidcontact zone.

Thus, for a lipolytic enzyme of the Humicola family, existing aminogroups may be removed, and 1-3 lysine residues may be introduced atpositions corresponding to the following amino acids in the Humicolalanuginosa lipase: 14, 15, 17-28, 35-42, 45, 54-65, 80-85, 87-95,110-116, 119, 144-151, 171-177, 195-209, 213-215, 219, 221-231, 234,238, 242-251, 257-269, particularly at position 199, 56, 27, 111, 118,37, 227, 226, 210, 95, 93, 255, 96, 252, 57 or 211.

Similarly, for a fungal cutinase, existing amino groups may be removedand 1-3 lysine residues may be introduced, e.g. by substitutionscorresponding to I5K, V158K, D63K, N44K and/or R149K. Examples areI5K+V158K+D63K and N44K+V158K+D63K.

Use of Modified Lipolytic Enzyme

The modified lipolytic enzyme can be used in any known application forsuch enzymes, e.g. in baking, in detergents or in immobilized form forvarious processes.

Baking

The modified lipolytic enzyme can be used in the preparation of dough,bread and cakes, e.g. to increase dough stability and dough handlingproperties, to increase the loaf volume or to improve the elasticity ofthe bread or cake. Thus, the enzyme can be used in a process for makingbread, comprising adding the enzyme to the ingredients of a dough,kneading the dough and baking the dough to make the bread. This can bedone in analogy with U.S. Pat. No. 4,567,046 (Kyowa Hakko), JP-A60-78529 (QP Corp.), JP-A 62-111629 (QP Corp.), JP-A 63-258528 (QPCorp.), EP 426211 (Unilever) or WO 99/53769 (Novo Nordisk).

Detergent

The lipolytic enzyme (e.g. a lipase) may be used as an additive in adetergent composition. This additive is conveniently formulated as anon-dusting granulate, a stabilized liquid, a slurry or a protectedenzyme. The additive may be prepared by methods known in the art.

Lipases tend to exert the best fat removing effect after more than onewash cycle (Gormsen et al., in Proceedings of the 3rd World Conferenceon Detergents, AOCS press, 1993., pp 198-203).

Immobilized Enzyme

The lipolytic enzyme may be immobilized by methods known in the art,e.g. by adsorption onto a polymer based carrier, by covalent binding toan activated polymer-based carrier (e.g. epoxy or aldehyde) and bygranulation, e.g. as described in WO 89/02916, WO 90/15868, WO 95/22606or WO 99/33964.

The immobilized lipolytic enzyme may be used for interesterification,e.g. of a water-insoluble carboxylic acid ester (such as a triglyceride)with another ester, with a free fatty acid or with an alcohol. Theimmobilized enzyme can also be used in ester synthesis or in resolutionof racemic compounds.

Detergent Composition

The detergent compositions of the invention may for example, beformulated as hand and machine laundry detergent compositions includinglaundry additive compositions and compositions suitable for use in thepretreatment of stained fabrics, rinse added fabric softenercompositions, and compositions for use in general household hard surfacecleaning operations and dishwashing operations.

The detergent composition of the invention comprises the lipase of theinvention and a surfactant. Additionally, it may optionally comprise abuilder, another enzyme, a suds suppresser, a softening agent, adye-transfer inhibiting agent and other components conventionally usedin detergents such as soil-suspending agents, soil-releasing agents,optical brighteners, abrasives, bactericides, tarnish inhibitors,coloring agents, and/or encapsulated or non-encapsulated perfumes.

The detergent composition according to the invention can be in liquid,paste, gels, bars or granular forms. The pH (measured in aqueoussolution at use concentration) will usually be neutral or alkaline, e.g.in the range of 7-11, particularly 9-11. Granular compositions accordingto the present invention can also be in “compact form”, i.e. they mayhave a relatively higher density than conventional granular detergents,i.e. form 550 to 950 g/l.

The lipase of the invention, or optionally another enzyme incorporatedin the detergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, particularly at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more particularly at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even moreparticularly at a level from 0.01% to 0.2% of enzyme protein by weightof the composition.

The detergent composition of the invention may comprise the lipase in anamount corresponding to 10-50,000 LU per gram of detergent, particularly20-5,000 LU/g, e.g. 100-1000 LU/g. The detergent may be dissolved inwater to produce a wash liquor containing lipolytic enzyme in an amountcorresponding to 25-15,000 LU per liter of wash liquor, particularly100-5000 LU/l, e.g. 300-2000 LU/l. The amount of lipase protein may be0.001-10 mg per gram of detergent or 0.001-100 mg per liter of washliquor.

More specifically, the lipase of the invention may be incorporated inthe detergent compositions described in WO 97/04079, WO 97/07202, WO97/41212, PCT/DK WO 98/08939 and WO 97/43375.

Surfactant System

The surfactant system may comprise nonionic, anionic, cationic,ampholytic, and/or zwitterionic surfactants. The surfactant system maycomprise a combination of anionic and non-ionic surfactant with 70-100%by weight of anionic surfactant and 0-30% by weight of nonionic,particularly 80-100% of anionic surfactant and 0-20% nonionic or 40-70%anionic and 30-60% non-ionic surfactant.

The surfactant is typically present at a level from 0.1% to 60% byweight, e.g. 1% to 40%, particularly 10-40%. particularly from about 3%to about 20% by weight. Some examples of surfactants are describedbelow.

Anionic Surfactants

Suitable anionic surfactants include alkyl sulfate, alkyl ethoxysulfate, linear alkyl benzene sulfonate and mixtures of these.

The alkyl sulfate surfactants are water soluble salts or acids of theformula ROSO₃M wherein R particularly is a C₁₀-C₂₄ hydrocarbyl,particularly an alkyl or hydroxyalkyl having a C₁₀-C₂₀ alkyl component,more particularly a C₁₂-C₁₈ alkyl or hydroxyalkyl, and M is H or acation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium),or ammonium or substituted ammonium.

Alkylbenzene sulfonates are suitable, especially linear (straight-chain)alkyl benzene sulfonates (LAS) wherein the alkyl group particularlycontains from 10 to 18 carbon atoms.

Suitable anionic surfactants include alkyl alkoxylated sulfates whichare water soluble salts or acids of the formula RO(A)_(m)SO₃M wherein Ris an unsubstituted C₁₀-C-₂₄ alkyl or hydroxyalkyl group having aC₁₀-C₂₄ alkyl component, particularly a C₁₂-C₂₀ alkyl or hydroxyalkyl,more particularly C₁₂-C₁₈ alkyl or hydroxyalkyl, A is an ethoxy orpropoxy unit, m is greater than zero, typically between about 0.5 andabout 6, more particularly between about 0.5 and about 3, and M is H ora cation which can be, for example, a metal cation (e.g., sodium,potassium, lithium, calcium, magnesium, etc.), ammonium orsubstituted-ammonium cation. Alkyl ethoxylated sulfates as well as alkylpropoxylated sulfates are contemplated herein. Specific examples ofsubstituted ammonium cations include methyl-, dimethyl,trimethyl-ammonium cations and quaternary ammonium cations such astetramethyl-ammonium and dimethyl piperdinium cations and those derivedfrom alkylamines such as ethylamine, diethylamine, triethylamine,mixtures thereof, and the like.

Other anionic surfactants include salts (including, for example, sodium,potassium, ammonium, and substituted ammonium salts such as mono- di-and triethanolamine salts) of soap, C₈-C₂₂ primary or secondaryalkanesulfonates, C₈-C₂₄ olefinsulfonates, sulfonated poly-carboxylicacids prepared by sulfonation of the pyrolyzed product of alkaline earthmetal citrates.

Nonionic Surfactant

The surfactant may comprise polyalkylene oxide (e.g. polyethylene oxide)condensates of alkyl phenols. The alkyl group may contain from about 6to about 14 carbon atoms, in a straight chain or branched-chain. Theethylene oxide may be present in an amount equal to from about 2 toabout 25 moles per mole of alkyl phenol.

The surfactant may also comprise condensation products of primary andsecondary aliphatic alcohols with about 1 to about 25 moles of ethyleneoxide. The alkyl chain of the aliphatic alcohol can either be straightor branched, and generally contains from about 8 to about 22 carbonatoms.

Further, the nonionic surfactant may comprise polyethylene oxidecondensates of alkyl phenols, condensation products of primary andsecondary aliphatic alcohols with from about 1 to about 25 moles ofethylene oxide, alkylpolysaccharides, and mixtures hereof, particularlyC₈-C₁₄ alkyl phenol ethoxylates having from 3 to 15 ethoxy groups andC₈-C₁₈ alcohol ethoxylates (particularly C₁₀ avg.) having from 2 to 10ethoxy groups, and mixtures thereof.

Examples of nonionic surfactants are alcohol ethoxylate, alcohol phenolethoxylate, polyhydroxy fatty acid amide, alkyl polyglucoside andmixtures of these.

EXAMPLES Example 1 Modified Lipases with an Average of 3 HydrophobicGroups

Modified lipases were prepared by covalently linking tetradecanoyl (C₁₄)and hexadecanoyl (C₁₆) groups, respectively, to Lipolase (Humicolalanuginosa lipase). Each lipase molecule has 7 amino groups(N-terminal+6 lysine residues), and it was estimated that an average of3 fatty acyl groups were linked to each molecule.

Example 2 Modified Lipases with 3 or 4 Hydrophobic Groups

Two variants of Lipolase were prepared by amino acid substitutions sothat the variants had the following amino groups. Other lysine residueswere substituted with arginine:

Three amino groups N-terminal and lysine at positions 46 and 98.

Four amino groups: N-terminal and lysine at positions 24, 46 and 98.

Fatty acyl groups (myristoyl and stearoyl, respectively) were linkedcovalently to the amino groups in each variant.

Example 3 Modified Lipases with 2 Hydrophobic Groups

A variant of Lipolase was prepared by substituting lysine residues witharginine to obtain a lipase variant having two amino groups, at theN-terminal and Lys 24.

Four different modified lipases were produced by linking the followinghydrophobic groups to the amino groups in the variant:

Stearoyl

C₁₈H₃₇—(O—CH₂—CH₂)₁₀₀

C₁₈H₃₇—(O—CH₂—CH₂)₂₁

Arachidoyl

A similar modified lipase may be made by linking to palmitoyl groups.

Example 4 Construction of Modified Lipases

Monopods, dipods and tripods are prepared from Lipolase by removing theN-terminal amino group by pyroglutamate cyclization and making variantsby amino acid substitutions having lysine at the following positions.Other lysine residues are substituted with arginine:

Monopod: lysine at position 98, 211 or 223.

Dipod: lysine residues at positions 98+233 or 96+255.

Tripod: Lysine residues at positions 24+98+223 or at positions57+96+252.

Hydrophobic groups (fatty acyl or polypropylene) are linked covalentlyto the lysine residues in each variant.

Example 5 First-Wash Performance

The two modified lipases were tested as described below, and unmodifiedLipolase was tested for comparison.

A number of variants according to the invention were tested in ananionic detergent. The experimental conditions were as follows:

-   -   Equipment: Thermostated Terg-o-tometer    -   Method: 1 cycle wash followed by line drying.    -   Wash liquor: 1000 ml per beaker    -   Swatches: 7 (cotton style # 400) swatches (9*9 cm) per beaker.    -   Stain: Lard coloured with Sudan red (0.75 mg Sudan red/g lard).        250 μl of lard/Sudan red heated to 70° C. is applied to the        center of each swatch, followed by line-drying over-night.    -   Water: 8.4° German hardness (° dH), Ca: Mg=2:1    -   Detergent: 1.8 g/l commercial detergent (Wisk)    -   Lipase dosage: as indicated below    -   Wash time: 20 min.    -   Temperature: 30° C.    -   Rinse: 15 minutes in running tap water.    -   Drying: Overnight at room temperature (˜20° C., 30-40% RH).    -   Evaluation: The reflectance was measured at 460 nm in a        reflectometer.        The results are given as ΔR (delta Reflectance)=reflectance of        swatches washed in detergent with lipase minus reflectance of        swatches washed in detergent without lipase.

Results:

Lipase Dosage, LU/I ΔR Reference Lipolase 1329 0.3 4011 0.7 InventionLipolase modified with C₁₄ 1617 1.9 4880 5.2 Lipolase modified with C₁₆1212 2.2 3658 5.5

The results clearly demonstrate that the modified lipases have animproved first-wash performance.

Example 6 Baking Tests

A chemically modified lipase was prepared by linking palmitoyl groups toHumicola lanuginosa lipase. The amounts of reagents were chosen so as tolink an average of 2-3 acyl groups to each lipase molecule.

The chemically modified lipase was compared the unmodified lipase in atraditional European straight dough baking procedure.

The volume and the shape of the rolls were evaluated. Volume wasevaluated by simple displacement of 10 rolls, and the shape wasevaluated by measuring height/width. The results were as follows

Invention Reference (modified lipase) (unmodified lipase) Lipase dosage,LU/kg flour 500 1000 Volume, ml/g 6.4 6.2 Shape (Height/width) 0.68 0.64

The results clearly show that the modified lipase at half the dosage ofthe reference has improved performance in terms of volume and shape.

1. A method of preparing a dough or a baked product from the dough whichcomprises adding to the dough a lipolytic enzyme composition comprisinglipolytic enzymes modified by covalently linking non-amino acidhydrophobic groups to an amino group, a thiol group, a hydroxyl group ora carboxyl group of the lipolytic enzymes, and wherein said hydrophobicgroups are present in said lipolytic enzyme composition on average oftwo to three hydrophobic groups per lipolytic enzyme.
 2. A doughcomposition comprising a lipolytic enzyme composition comprisinglipolytic enzymes modified by covalently linking non-amino acidhydrophobic groups to an amino group, a thiol group, a hydroxyl group ora carboxyl group of the lipolytic enzymes, and wherein said hydrophobicgroups are present in said lipolytic enzyme composition on average oftwo to three hydrophobic groups per lipolytic enzyme.
 3. The method ofclaim 1, wherein the hydrophobic groups of the lipolytic enzymecomposition are fatty acyl groups.
 4. The method of claim 1, wherein thelipolytic enzymes consist of two or three amino acids having an aminogroup and wherein said hydrophobic groups are covalently linked to thetwo or three amino acids.
 5. The method of claim 1, wherein thehydrophobic groups are selected from the group consisting of a fattyacyl group, a polyalkoxy and an alkyl-polyalkoxy group.
 6. The method ofclaim 1, wherein the lipolytic enzymes are Humicola lipolytic enzymes.7. The method of claim 1, wherein the lipolytic enzymes are Humicolalanuginosa lipases.
 8. The method of claim 1, wherein the lipolyticenzymes are selected from the group consisting of a lipase, a cutinaseand a phospholipase.
 9. The method of claim 1, wherein a lipolyticenzyme composition comprises lipolytic enzymes modified by covalentlylinking non-amino acid hydrophobic groups to an amino group of thelipolytic enzymes.
 10. The method of claim 1, wherein a lipolytic enzymecomposition comprises lipolytic enzymes modified by covalently linkingnon-amino acid hydrophobic groups to a thiol group of the lipolyticenzymes.
 11. The method of claim 1, wherein a lipolytic enzymecomposition comprises lipolytic enzymes modified by covalently linkingnon-amino acid hydrophobic groups to a hydroxyl group of the lipolyticenzymes.
 12. The method of claim 1, wherein a lipolytic enzymecomposition comprises lipolytic enzymes modified by covalently linkingnon-amino acid hydrophobic groups to a carboxyl group of the lipolyticenzymes.